Project Summary/Abstract The goal of this project is to develop a platform that will allow cells to be sorted based on properties determined by microscopic imaging. While morphology-based cell cytometers and flow cytometers exist, these instruments do not permit separation of cells by their morphological properties. Combining a ?Sorting Microscope? with CRISPR/Cas9-based mutagenesis will enable in vivo or in vitro forward genetic screens to identify genetic programs that regulate single cell phenotypes such as shape, intracellular organization, and protein or organelle subcellular localization. We will perform a proof-of-concept study in which we use the Sorting Microscope to perform a forward genetic screen based on cell morphology. Cardiomyocyte form has evolved to precisely fulfill their functional role. Among these structural adaptations, perhaps the most striking are the transverse tubules (T-tubules), a network of tubular invaginations of the plasma membrane that penetrate into the center of the cardiomyocyte. T-tubules are considered a hallmark of mature CMs and are required for efficient excitation-contraction coupling, yet little is known about the factors that regulate T-tubule formation. Using our platform for in vivo CRISPR/Cas9-based somatic mutagenesis and the Sorting Microscope, we will undertake a proof-of-concept forward genetic screen to identify genes required for T-tubule formation. Our Specific Aim is to establish the technology and methodology to perform forward genetic screens based on cell morphology. In Specific Aim 1.A, we will develop a platform for automated identification, labeling, and sorting of cells by morphology. This open-source hardware and software platform will be designed to facilitate widespread dissemination. In Specific Aim 1.B, we will use the hardware/software platform to identify genes required for T-tubule formation in cardiomyocytes. To achieve these goals, we have assembled an interdisciplinary team consisting of a bioengineering group (Voldman) and a cardiac biology group (Pu). Voldman?s bioengineering group has expertise in image analysis, automation, and in morphology-based cell separation. The Pu lab has expertise in Cas9-based in vivo somatic mutagenesis and in cardiac biology. We anticipate that the resulting technology will enable the power of forward genetics to be unleashed on diverse problems in developmental biology, with direct relevance to human diseases.